2beta-hydroxy-9 alpha-halo-11-oxygenated pregnenes



Unite Stas amass) Patented Nov. 13, 1952 This invention relates to new steroid compounds. More particularly it relates to 2,2l-dihydroxy-4-pregnene 3,20- diketones and esters thereof and method of preparing the same.

Under the fermentation conditions as described by Fried et al., in their US. Patents 2,709,705; 2,855,343; 2,855,410 using Strcptomyces roseochromogenus, (WaksmanNo. 3689) a product hydroxylated in the 16a-position is obtained from many steroid substrates. The 90cfiuorohydrocortisone 16a-hydroxyl derivative is described in J. Am. Chem. Soc. 79, 4818 (1957).

We have found that when different carbohydrate energy sources are used, with a selected strain Streptomyccs roseochromogenus (ATCC 3347) the presence of other steroidal products was detected via paper chromatograms. One of these other products has the same order of mobility as the l6a-hydroxylated product, but actually is about 90% as mobile in carefully conducted experiments, so that the presence of the second component can be proven. Chemical studies on the new steroids have been made, and the conclusion drawn that the 9mfiuorohydrocortisone substrate is hydroxylated at a new position, namely, the 2/3-position, rather than the 16aposition, in the case of the steroid with 90% of the mobility of 16a-hydroxy-9u-fluorohydrocortisone, and that a steroid with 34% of the mobility of 16oc-hYd1OXY-9ctfluorohydrocortisone is the 2B,l6ot-dihydroxy-9a-fluorohydrocortisone.

The principal distinction between fermentations which give 2,8-hydroxylation and which do not give 2,8-hydroxylation of steroid substrates appears to be a matter of selection of strains of S. roseochromogemis although medium composition may play a minor role. Under the conditions of the present invention S. roseochromogenus (ATCC 3347) will give 2,3-hydroxylated steroids in up to fifty percent yields.

The fermentation conditions to prepare the Zfi-hydroxy pregnenes of the present invention such as, for example, 2,8-hydroxy-9a-fiuorohydrocortisone are similar to those described in the prior art with exceptions noted above. The ZB-hydroxylation takes place as the major product when S. roseochromogenus (ATCC 3347) is used in medium such as, for example, that described in US. Patent 2,855,343.

The isolation of these new 2 8-hydroxylated steroids is accomplished by the usual means of solvent extraction of the harvest broth, concentration of the extract, and re-.

covery of the crude steroid crystals. The separation of the crude steroid mixture can best be accomplished by the use of aqueous sodium borate solutions to remove by complex formation those steroids hydroxylated in the 160:- position. Thus with a solvent solution of the mixed steroids, 16u-hydroxy-9a-tluorohydrocortisone is extracted into aqueous borate solution and unaltered 9zx-fluorohydrocortisone is also retained in the solvent extract; 25,160:- dlhYdlOXY-9oc-fitl0rOllYdIOCOIfiSOIlfi is also extracted, While 2 3-hydroxy-9u-fluorohydrocortisone remains in the organic solvent extract. Thus, the organic solvent extract may be freed of those steroids containing the 1606,1706- cis-diol feature. The so purified solvent extract is then concentrated and subjected to chromatography for the final separation of the desired ZB-hydroxypregneues.

The Zfl-hydroxypregnenes which have present cis- 16a,17 t-dihydroxy groups react with alkanones and alkanals to produce the corresponding substituted methylenedioxy pregnenes as shown hereinafter in the examples. These same Zfi-hydroxylated cis-16a,l7a-dihydroxy pregnanes undergo D-homoannulation in the same manner as already known for triamcinolone, and illustrated in Example XXII.

The 2B-hydroxypregnenes such as 2,6-hydroxy-9a-fiuorohydrocortisone, 213,16 a-dihydroxy-9u-fluorohydrocortisone are active glucocorticoids, while the acetonide type of derivatives of the latter compound is highly active topically.

The following examples illustrate the preparation of the compounds of the present invention.

EXAMPLE I Preparation of 9a-Flu0r0-2BJ15,Z7x,21-Tetrahydr0xy- 4-Pregnene-3,20-Di0ne A fermentation medium composed of cornsteep liquor, 20 g./liter; calcium carbonate, 7 g./l.; ammonium sulfate, 2 g./l.; dipotassium hydrogen phosphate, 3 g./l.; lard oil, 0.2% by volume; using glycerine as carbohydrate source, 30 g./l.; is prepared and inoculated with a 24-hour vegetative growth of S. roseochromogenus (ATCC 3347). Fermentation is carried out in 250 ml. Erlenmeyer flasks containing 50 ml. of the sterilized medium. After a 24-hour growth period the culture is charged with 9a-iluorohydrocortisone (9a-fluoro-11B,17a,21-trihydroxy 4 pregnene- 3,20-dione) dissolved in dimethylformamide, so that the final solvent concentration in the culture is less than 1% by volume and the final steroid concentration is 500 u.g./ml. The culture is aerated by shaking on a rotary shaker at 185 rpm. with a two inch diameter throw. The temperature is 26.5 C. After 72 hours of fermentation, the culture is sampled for analysis. A total steroid analysis by polarography indicates 360 ,ug./ml. of products and unaltered substrate. Paper chromatography indicates that both unaltered substrate and the desired 2fi-hydroxy product, 9a fiuoro 2B hydroxyhydrocortisone (c fiuoro- 2 3,11fi,17a,21-tetrahydroxy 4 pregnene-3,20-dione) are present together with the l6a-hydroxylated product 9afiuoro 16a hydroxyhydrocortisone (9ot-fluoro-1l/3,16a, 17u,21-tetrahydroxy-4-pregnene-3,ZO-dione) EXAMPLE II Preparation of 9a-Flu0r0-2fl-Hydroxyhydrocortisone A fermentation medium essentially the same as that described in Example I except that the amount of glycerine utilized is varied between 40 g./l. and 60 g./l. is used. S. roseochromogenus (ATCC 3347) is used for fermentation with 9u-fiuorohydrocortisone as the substrate (added in the same manner as described in Example I). After 72 hours of aerated fermentation, the broth is sampled for analysis. Paper chromatograms indicates that unaltered substrate is present together with the l6ot-hydroxy product (9o-fiuoro-16x-hydroxyhydrocortisone) and the Zfl-hydroxylated pro-duct (9a-fiuoro-2B- hydroxyhydrocortisone). The following proportions are observed on analysis of each fermentation broth:

3 EXAMPLE III Preparation of 9a-Fluoro-2fl,11;8,16a,1 7a21-Pentahydroxy l-Pmgnene-iZO-Dione T o a growing culture of S. roseochromogertus (ATCC 3347) prepared as described in Example I, using glycerine as the carbohydrate source, is added 9a-fiuoro-16aydroxy-hydrocortisone in solution so that the final steroid concentration is 500 reg/ml. After 72 hours of fermentation, paper chromatographic analysis showed the presence of unaltered substrate and the presence of 9a-fiuoro- 25,160 dihydroxyhydrocortisone (9ot-fltl01'02[3,11fi,16a, 17a,2 1-pentahydroxy-4-pre gnene-3,20-dione).

EXAMPLE IV Preparation of 9a-FIL Lor0-2,8,16a-Dihyriroxyhydrocortisone Preparation of 9a-Fluor0-2fl-Hydroxyhydrocortisone The fermentation broth from a S. roseochromogenus (ATCC 3347-) fermentation performed as described in Example I is acidified to pH 4.0 with 25% sulfuric acid and the acidified broth filtered through a diatomaceous earth filter pad to remove micelial solids. The clear filtrate is adjusted to pH 7 with 10% sodium carbonate solution and is then extracted with an equal volume of methyl isobutyl ketone. A second extraction is performed and the solvent extracts are combined and concentrated under diminished pressure to a point of incipient crystallization (ca. 1% of the original volume). The crystals so obtained are filtered and consist of 9a-fiuoro-l6a-hydroxyhydrocortisone, 9a-fluQro-Zfihydroxyhydrocortisone and 9a fiuoro-2fi,l6u-dihydroxyhydrocortisone, together with traces of other fermentation impurities. Further concentrationof the mother liquors yields a secondcrop of solids, composed essentially of the same steroids as is the first crystal crop, although some unaltered substrate maybe. present in the second crops.

Recrystallization of the first crop or of the first and second crops combined from a suitable solvent such as alcohol yields the purified 9ct-flt101'0-l6oc-hYdlOXYhYdI'O- cortisone and depending on the exact composition of the crude crystalline product obtained from each fermentation, the 9 t-fluoro-l6a-hydroxyhydrocortisone so produced is either freefrom Zfi-hydroxylated steroids or may stillbe contaminated with traces of the 213-hydroxylated steroids, thus requiring further purification operations.

EXAMPLE VI Separatipn of 25- Hydrxylated Products From 160;-

H ydroxy lated Products The .mother liquors remaining after the first two or three crops of solid 16a-hydroxy-9a-fluorohydrocortisone had been recovered are quite rich proportionately in the ZB-hydroxylated pro-duct. The mother liquor is very viscous at this stage, and is diluted with fresh methyl isobutyl ketone for analysis and further separations. A volume of 150 m1. of the mother liquor concentrate obtained as described in Example V, and assaying .to contain about 20% by weight of polarographically active steroidal components, is diluted with 300 ml. of methyl isobutyl ketone, and then extracted five times with 600 ml. portions of aqueous sodium tetraborate solution. The fully extracted methyl isobutyl ketone solution is thus free from l6a,17a-diols of the type 9ot-fl1101'O-llfi,l6ot,17a,2ltetrahydroxy-4-pregnene-3,ZO-dione, etc. as evidenced by paper chromatographic analysis. The several borate extracts are further washed several times with fresh methyl isobutyl ketone to recover small amounts of desired product, and the combined methyl isobutyl ketone solution rich in desired steroid together-with the methyl isobutyl ketone washes of the spent borate extracts are concentrated to dryness under reduced pressure. The residue so obtained is analyzed by paper chromatography and found to contain large amounts of the desired steroid, not contaminated with either 9a-fluoro-1 lfi,16a,17 x,21-tetrahydroxy-4-pregnene-3,20-dione or with 9ot-fill01'O-213,11, 16a,l7a,2l-pentahydroxy-4-pregnene-3,20-dione.

EXAMPLE VII Isolation of 2fi-Hydroxy-Qa-Fluorohydrocortisone F luoro 25,1 15,1 7a-21 -Tetrahydroxyi-Pregnene-3,20- Dione) A solution of the desired ZB-hydroxy steroid which has been previously extracted with dilute borate solution according to the Example VI to remove the l6a,'l7a-diols (9a-fluoro-11t3, 16a, 17a,2l-tetrahydroxy-4-pregnene-3, 20-dione and 9a-fluoro-2B,l15,16 17a21-pentahydroxy- 4-pregnene 3,20-dione) is analyzed by papergram to insure that the 16st, 17rx-diOlS have been properly removed. Such a solution, 780 ml., of desired steroid in methyl isobutyl ketone is treated with 80 g. of activated silica gel, stirred, and concentrated under reduced pressure to dryness to yield a guru adsorbed on silica gel. The adsorbed steroid/ silica gel mixture is slurried several times with benzene and concentrated to dryness under reduced pressure each time, to insure the complete removal of methyl isobutyl ketone solvent. The thusprepared charge of adsorbed steroid/silica gel is slurried in benzene and placed on top of a silica gel column (prepared in benzene, a three-inch column, charged with 950 g. of silica gel slurried in benzene). Elution of the column with benzene, with benzene/ether mixtures, and with pure ether removes unwanted impurities. Then elution with ether/chloroform (:5, volume/volume) yields a fraction rich in the desired steroid component. After concentration of the eluates to dryness a residue is obtained which is crystallized from methanol, yielding crystals, homogeneous on papergrams as the desired steroid. After recrystallization from methanol, including a charcoal treatment, and from acetone, the product, MP. 244-248, was recovered. Further purification yielded pure product, M.P. 245-249", [(11 +l.5 (methanol),

258? 239 n tta... 352) homogeneous on papergrams, and exhibiting infrarel adsorption spectra in KBr disks consistent with the assigned structure.

EXAMPLE VIII Preparation of 9et-Flu0ro-2BJoa-Dihydroxyhydrocortisone To a 24-hour vegetative growth of S. roseochromogenus (ATCC 3347) grown in 30 l. of medium containing corn steep liquor (750 g.), starch (1200 g.), calcium carbonate (150 g.), dipotassium hydrogen phosphate (30 g.) and lard oil (60 ml.) is added a solution of 7.5 g. of 9a-fiuorohydrocortisone dissolved in ml. of dimethylformarnide. Fermentation is continued for 88 hours, at which time the'total steroid content of the broth assayed 132 ,ug/ml. Paper chromatographic analysis indicates that the content of the desired 9::- fiuoro-Zfi,l6a-dihydroxyhydrocortisone is l00,ug./ml. The harvested broth is, adjusted to pH 4.3 with 50% sulfuric acid, slurried with 2360 g. of diatomaceous earth filter aid, and filtered. The clear filtrate is reslurried with 30 l. of water at pH 3.6 and refiltered. The combined filtrates are neutralized to pH 6.4 with 20% sodium carbonate solution, then extracted three times with 25 1. portions of methyl isobutyl ketone. The pooled extracts are concentrated to a residue under reduced pressure, the residue is dissolved in 500 ml. of methanol, and analyzed chromatographically. The major steroidal component found is the desired 9a-fluoro-2B,16a-dihydroxyhydrocortisone. The methanol solution is evaporated, the solids so obtained are slurried in acetone/ethyl acetate, the undissolved solids then recrystallized from pyridine, from ethyl acetate, finally from acetone and water, yieldin crystalline 9a-fiuoro-2 3,loa-dihydroxyhydrocortisone free from other steroids. After several other recrystallizations from acetone, the material melted 220-223" C., and is characterized by [ab -26.5; A max. in ethanol 238 me with e 14,700.

EXAMPLE IX Preparation of 9cc-FlLl0r0-16a,1 7a-lsopropylidenedioxy- 2 5,1 1 3,21 -Trilzydroxy-4-Pregnene-3,20-Dione One hundred milligrams of 9a-fiuoro-2fi,llfi,l6a,l7a, 21-pentahydroxy-4-pregnene-3,20-dione is suspended in '5 ml. of acetone to which suspension are added several drops of 70% perchloric acid. The steroid rapidly dissolves in the acetone. The perchloric acid is neutralized with saturated aqueous sodium bicarbonate solution, the excess acetone removed by distillation in a vacuum, and the precipitated steroidal derivative is recovered by filtration. The crude acetonide may be crystallized from acetone/petroleum ether. The acetonide derivative exhibited infrared bands at 290p consistent with the presence of three hydroxyl groups in the molecule (25, 115, and 21), 338 5.80 11. (the 20-carbonyl group), 592 (the A -3-carbonyl group, 6.10 (the A -group), 6.85 r, 7.20 (C-methyl) 8.15;t(CO-C), 9.40 11.20,, and 11.60,a. The mobility of the steroid on papergrams is less than that of the non-2,8-hydroxylated analog.

EXAMPLE X Preparation of 25,21-Diacetxy-1 1/3,] 7oc-Dihydr0xy-9u- F luoro-4-Pregnene-3,20-Dione One hundred milligrams of 9a-fluoro-2/3,11;3,l7u,21- tetrahydroxy-4-pregnene-3,20-dione, as obtained in Example VII is dissolved in 10 ml. of dry pyridine, and 0.1 ml. of acetic anhydride is added. After standing at room temperature over-night, the reaction mixture is quenched with excess methanol, and the solvents are removed under reduced pressure. Additional methanol is added and the preparation concentrated under reduced pressure until the odor of pyridine is removed, at which time the steroidal residue is dried well under reduced pressure. The residue may be crystallized from acetone/ petroleum ether to yield fine needles, M.P. 1325-1405 (Kofier); [111 +481 (methanol); and homogeneous on paper chromatograms. The diacetate exhibits infrared adsorption bands at 290p (consistent with the presence of two hydroxyl groups, at 2/3- and at 170:), 3.41 6.73m, 5.78 1. (20-carbonyl and acetate carbonyl groups), 5.95 1. (A -ketone), 6.15;; (A -group), 7.29 1, 8.15;, 954a, and 1132a.

EXAMPLE XI Preparation of 2 5,21 -Diacet0xy-9a-F luoro-I 70,H ydroxy- 4-Pregnene 3,11,20-Trione To a solution of 150 mg. of chromic anhydride in 15 ml. of dry pyridine is added a solution of 250 mg. of 2,8,21-diacetoxy-1 1 3,17a-dihydroxy-9a-fluoro-4-pregnene- 3,20-dione in 20 ml. of pyridine. After 20 hours the solution is poured into water containing 0.7 g. of sodium bicarbonate. The mixture is steam distilled to remove pyridine, and then extracted with ethyl acetate. The extracts are washed well with water, then dried and concentrated under reduced pressure. The residue so obtained is crystallized from acetone/petroleum ether.

Several recrystallizations are required to obtain the pure trione, homogeneous on paper chromatograms.

EXAMPLE XII Preparation of 9a-Chlor0-2f3,1 15,1 7a,21-Tetrahya'r0xy- 4-Pregnene-3,20-Di0ne Under fermentation conditions as described in Example II, using S. roseochromogenus and with glycerine as carbohydrate source, 9a-chloro-11B,17a,21-trihydr0xy-4-pregnene-3,20-dione is converted to the ZB-hydroxy derivative, together with the 16a-hydroxy derivative, as per Example III. Papergram analyses permit determination of when to terminate the fermentation in such a manner as to yield optimum amounts of the 2 3-hydroxy-9a-chloro-hydro, cortisone. Isolation of the products and separation of the products in the same way as described in Examples V, VI, and VII gives pure crystalline 2,8-hydroxy-9a-chlorohydrocortisone.

EXAMPLE XIII Fermentation of 16a-hydroxy-9a-chlorohydrocortisone or of'2B-hydroxy-9tx-chloro-hydro-cortisone with S. roseochromogenus under conditions described in Examples I, II, and III yields as a major product the 2,8,16a-dihydroxylated products, namely, 213,16a-dihydroxy-9u-chlorohydrocortisone, which is indicated as a polar spot on paper chromatograms. The polar steroid spot is in the same vicinity as that of 2,8-l6oc-dihYClIOXY-9zz-fll10l'0hYdlOCO1ilsone and is positive to alkaline tetrazolium blue reagent.

EXAMPLE XIV In a medium consisting of soybean oil meal, 20 g./l.'; glucose, 40 g./l.; calcium carbonate, 2.5 g./l.; dipotas sium hydrogen phosphate, 0.4 g./l.; and soybean oil, 2 1111/1. the Waksman strain of S. roseochromogenus (No. 3689) is grown, and 9a-fluorohydrocortisone is added as a solution in dimethylformamide so that the final steroid concentration is 500 ag/ml. Aerated fermentation is conducted for 96 hours, a sample being withdrawn at 72 hours. At 72 hours 32% of unaltered substrate remained, at 96 hours 26%. Paper chromatographic analysis indicates that the major steroid product is 9a-fiuoro-16a-hydroxyhydrocortisone, with no 9a-fluoro-2fi-hydroxy-hydrocortisone present.

EXAMPLE XV Fermentation conditions exactly as described in Example XIV are carried out, except that the fermentation is continued to 144 hours. Analysis at that time indicates no unaltered substrate, 84% of 9a-fluoro-16a-hydroxyhydrocortisone, with no detectable ZB-hydroxylated product.

EXAMPLE XVII Fermentation conditions employing a medium composition of corn steep liquor, 25 g./l.; starch, 40 g./l.; calcium carbonate, 5 g./l.; and lard oil, 2 ml./l. and subtrate of 9a-fluoro-hydrocortisone with the S. roseochromogenus Waksman strain indicates no 2,8-hydroxylation has occurred after 72 hours of aerated fermentation.

EXAMPLE XVIII Using the medium composition of Example XVII but employing the selected strain (ATCC 3347) the products 7 of the fermentation are 62% of 90t-flllOl0-16ot-11YdIOXY- hydrocortisone and 38% of the 2e-hydroxy product, 904- fluoro-Zfl,16a-dihydroxyhydrocortisone.

EXAMPLE XIX Using fermentation conditions exactly as described in Example XVIII a series of samples are withdrawn from the fermenting brothand analyzed for 2,8-hydroxylated species. Bot-h 9a-fluoro-Zp-hydroxyhydrooortisone and 9a-fluoro-25,16tt-dihydroxyhydrocortisone are analyzed, as Well as the non-Zfl-hydroxylated steroid 9a-fluoro-16tz-hydroxyhydrocortisone.

2B-HYDROXYLAIED STEROIDS In a medium described in Example XI of U.S. Patent No. 2,855,343 consisting of soybean meal, 15 g./1.; glucose g./l.; soybean oil, 2.2 g./l.; calcium carbonate, 2.5 .g./l.; with the pH adjusted to pH 70 the Waksman strain No. 3689 of S. roseochromogenus is grown under aerobic conditions. '9m-fluorohydrocortisone is added as a solution in ethanol so that the final steroid concentration is 330 ,ug./ml., and the fermentation continued for 72 hours. At this time about 38% of the unaltered substrate is left, 46% of the product 9m-fluoro-16a-hydroxyhydrocortisoneis present, but no detectable amount of '2B-hydroxylated steroids could be-found by chromato raphic means.

EXAMPLE XXI In a medium described in EXample'XVII of U.S. Patent 2,855,343 consisting of soybean oil, 8.8 g./l.; soybean meal 30 g./l.; cobalt nitrate dihydrate, 5 mg./l., the Wakeman strain No. 3689 of S. roseochromogenus is grown. A solution of 9ut-fluorohydrocortisone in ethanol is added so that the final steroid concentration is 330 g/ml. and fermentation is continued for 72 hours, at which time chromatographic analysis indicated that 35% of the substrate remains unaltered. The product 9a fluoro-16a-hy- 'dIOXYhYdIOCOI-iiSOHC accounted for 50% of the steroids found, and no detectable Zli-hydroiylated products could be found.

EXAMPLE XXII 'Five hundred milligrams of 9a-fluoro-2/8,16a-dihydroxyhydrocortisone is dissolved in rnethanol and an equal volume of a 1% solution of sodium in methanol is added. After standing for fifteen minutes the solution is neutralized with acetic acid, and evaporated to dryness. After washing the residue with water, the insolubles are dissolved in alcohol, treated with decolorizing charcoal, and precipitated by the addition of ten-volumes of water. The solids are chromatographed on diatomaceous earth using the solvent system dioxane/cyclohexane/water in whereinR is selected from the group consisting of hydrogen and lower alkanoyl radicals and R and R are lower alkyl radicals.

References Cited in the file of this patent UNITED STATES PATENTS 2,280,828 Inhofien Apr. 28, 1942 2,602,803 Kaufmann et a1 July 8, 1952 2,666,016 Hechter et al. Ian. 12, 1954 2,756,179 Fried et al July 24, 1956 2,773,058 Bernstein et a1 Dec. 4, 1956 2,777,864 Bernstein et a1 Jan. 15, 1957 2,910,487 Julian et a1 Oct. 27, 1959 2,968,595 Greenspan et al I an. 17, 1961 

2. A COMPOUND OF THE FORMULA: 